Double salts of fumaric acid with a carnitine and an amino acid and food supplements, dietary supplements and drugs containing same

ABSTRACT

Double fumarates of L-carnitine or isovaleryl L-carnitine and an amino acid having formula (I) are disclosed which are useful as active ingredients of food supplements, dietary supplements or drugs.                    
     wherein: R is hydrogen or isovaleryl; and 
     [A + ] is a positively charged amino acid selected from the group consisting of creatine, ornithine, lysine, arginine and histidine.

This application is the US national phase of international applicationPCT/IT01/00199 filed Apr. 24, 2001, which designated the US.

The present invention relates to stable and non-hygroscopic double saltsof fumaric acid (hereinbelow “double fumarates”) with a “carnitine”,wherein by “carnitine” either L-carnitine or isovaleryl L-carnitine aremeant and an amino acid. The invention also relates to food supplements,dietary supplements, nutraceuticals and drugs containing said doublefumarates.

Fumaric acid [(E)-2-butenedioic acid] exhibits interesting applicationsin both the nutritional and therapeutical field.

It is used as substitute for both tartaric acid which may bring aboutunpleasant gastrointestinal side effects due to its laxative effects, inthe preparation of beverages and baking powders, and citric acid infruit drinks.

The cardioprotective effect of fumaric acid has been assessed in theperfused rat heart (La Plante et al. “Effects and metabolism of fumaratein the perfused rat heart. A ¹³C mass isotopomer study”, Am. J. Physiol.272: E74-E82, 1997) and in the immature myocardium (Pearl et al.“Fumarate enriched blood cardioplegia results in complete functionalrecovery of immature myocardium” Ann. Thorac. Surg. 57: 1636-41, 1993).

Furthermore, fumaric acid is a “pharmacologically acceptable acid”: itssalts are in fact encompassed in the list of “FDA-approved commerciallymarketed salts” published e.g. in the Journal of PharmaceuticalSciences, Vol. 66, No. 1, (1977) pages 1-19.

Conversion of drugs to their respective pharmacologically acceptablesalt forms is a widely utilized means for optimizing the administrationforms or certain properties of the drugs, such as stability,hygroscopicity, flowability and the like.

Both L-carnitine acid fumarate and isovaleryl acid fumarate are knowncompounds. (Fumaric acid is a dicarboxylic acid: in the aforesaid acidfumarates only one of the two carboxylic groups is salified).

L-carnitine acid fumarate, whose preparation and physico-chemicalproperties are disclosed e.g. in U.S. Pat. No. 4,602,039, has beendeveloped in order to overcome the complex problems of storage andprocessing due to L-carnitine inner salt hygroscopicity. L-carnitineacid fumarate is in fact very stable and, without provokinggastrointestinal side effects, shows a profile of humidity resistanceeven better than that of L-carnitine tartrate, a further non-hygroscopicsalt which was also developed to overcome L-carnitine hygroscopicity.

The tartrate, however, has the advantage that both its carboxylic groupsare salified with L-carnitine and consequently it contains a higherpercentage in L-carnitine (68% vs. 58%).

Also isovaleryl L-carnitine acid fumarate, whose preparation isdisclosed in the U.S. Pat. No. 5,227,518, is a stable compound endowedwith considerable resistance to humidity.

Every endeavour made to salify also the free carboxylic group of acidfumarates wherein the other carboxylic group is salified withL-carnitine or isovaleryl L-carnitine has failed to-date. For instance,the attempt of preparing L-carnitine fumarate (i.e. the neutral saltwhich would have the advantage of a very high percentage in L-carnitine,about 73.5% vs. 68% in the tartrate and 58% in the acid fumarate)results in a highly hygroscopic substance which likely consists of amixture of L-carnitine acid fumarate and L-carnitine inner salt. It isthis latter which imparts high hygroscopicity to the end product as awhole.

Similar failures occur if the attempt is made to salify the freecarboxylic group of acid fumarates with alkanoyl L-carnitines, such asacetyl and propionyl L-carnitines.

It is an object of the present invention to provide stable,non-hygroscopic double fumarates wherein one of the carboxylic groups offumaric acid is salified with either L-carnitine or isovalerylL-carnitine and the other carboxylic group is also salified with acompound endowed with useful nutritional, dietary or therapeuticalproperties.

It is, therefore, apparent that the utility of the salts of the presentinvention is to be found not only in their lack of hygroscopicity andhigh stability, but also insofar as both their cationic moietiescontribute to the nutritional, energetic and/or therapeutic efficacy ofthe salt as a whole. The aforesaid efficacy of these novel salt is,therefore, not to be attributed exclusively to the “carnitine” moiety ofthe salt.

The aforesaid object is achieved by the double fumarates of L-carnitineor isovaleryl L-carnitine and an amino acid having the formula (I):

wherein: R is hydrogen or isovaleryl; and

[A+] is a positively charged amino acid selected from the groupconsisting of creatine, ornithine, lysine, arginine and hystidine.

The following compounds of formula (I) are to be considered specificallyencompassed by the present invention:

L-carnitine and creatine fumarate;

isovaleryl L-carnitine and creatine fumarate;

L-carnitine and ornithine fumarate;

isovaleryl L-carnitine and ornithine fumarate;

L-carnitine and lysine fumarate;

isovaleryl L-carnitine and lysine fumarate;

L-carnitine and arginine fumarate;

isovaleryl L-carnitine and arginine fumarate;

L-carnitine and hystidine fumarate; and

isovaleryl L-carnitine and hystidine fumarate.

Lysine, arginine and hystidine are amino acids occurring in proteins,i.e. they are three out of the twenty amino acids which are obtained viacontrolled hydrolysis of naturally occurring proteins (see, e.g., J.David Rawn, Biochemistry, Chapter 3 “Amino acids and the primarystructure of proteins”; McGraw-Hill, 1990).

Whilst in order to illustrate the nutritional and therapeutic efficacyof the amino acids in general reference is made to the conspicuouslyvast literature published to-date on this matter (see, e.g., F. Fidanzaand G. Liguori, Nutrizione umana, Chapter 3: “Le proteine”, CasaEditrice Libraria Idelson, 1995; and I. Goldberg (Ed.), FunctionalFoods, Chapter 12, “Amino acids, peptides and proteins” Chapman & Hall,Inc. 1994), it is deemed useful to briefly address the topic of creatineand ornithine in view of their peculiar physiological role.

Creatine is an amino acid present in considerable amounts in theskeletal muscle tissue of vertebrates wherein about ⅔ thereof occurs ascreatine phosphate.

Creatine is biosynthesized mainly in the liver and kidneys from threeamino acids: glycine which provides the carbon skeleton, arginine whichreleases the amidino group and methionine which releases the methylgroup. Creatine is excreted with urine as creatinine. Creatine can betaken with the diet since it is principally present in meat. However, inorder to take 10 grams/day of creatine, 2.5 kg of meat should be eaten.The exogenous supply and endogenous biosynthesis must compensate for thedaily turn-over of creatine to creatinine which in a 70-kg male subjectcan be estimated at about two grams.

The physiologic role of creatine is extremely important: principally inthe skeletal muscle, but in the brain, liver and kidneys as well,creatine—by reversibly taking up ATP's phosphate groups—plays the roleof reservoir of the energy-rich phosphate radicals. This reaction iscritically important since ATP can not be stored in tissues in excess ofa very limited threshold. It is creatine phosphate whose content intissues is five times as much that of ATP, which provides for phosphategroups supply. Following a moderately wearying physical exertion, thecreatine phosphate present in the skeletal muscle decreases in a farrelevant amount than ATP does, thus showing that creatine phosphaterephosphorilates ADP as ATP becomes dephospharilated.

When the rate of ATP's metabolic production exceeds ATP's utilization,this results in creatine phosphate formation. Creatine phosphate is,therefore, a reservoir of immediately available energy, suitable forcounterbalancing energy demands exceeding ATP's synthesis rate inmetabolic phosphorylation processes.

Creatine is mainly taken by athletes and sportsman insofar as itincreases the skeletal musculature if its intake is accompanied bylasting physical exertion. Creatine intake results in a lowering of fatwhile it enhances skeletal muscle. Recent researches have shown that thecombined intake of creatine and carbohydrates enhances creatine effectsowing to insuline production that is stimulated by simple sugars whichlikely play a role in creatine exportation to muscle cells.

Ornithine, a non-proteogenic amino acid, is a lower homolog of lysineand an important intermediate in urea biosynthesis cycle wherein it issynthesized by arginine transguanidinization. Ornithine can also beconverted to glutamic acid.

The fumarates of formula (I) fully accomplish the object of the presentinvention insofar as they are not only stable, non-hygroscopic compoundswhich favourably lend themselves to the preparation of solidpresentation forms which are the preferred ones for nutraceuticals andnutritional and dietary supplements, but also combine in single saltsthe complementary physiological activities of a “carnitine” and of theaforesaid amino acids.

For instance, those fumarates which combine creatine and a “carnitine”synergistically in a single compound, stimulate on one hand theproduction of energy in the myocytes, particularly in type I musclefibers, allowing important energy-carriers (the fatty acids) to enterthe mitochondrion, and, on the other hand, stimulate the ATP formed viacellular respiration (oxidative phosphorilation) to leave the sameorganelle. The ATP provides the mechanical energy needed for musclecontraction.

The following non-limiting examples illustrate the preparation andphysico-chemical properties of some compounds of the present invention.

EXAMPLE 1 L-Carnitine and Creatine Fumarate (BS/231)

14.9 g (0.1 moles) of creatine monohydrate and 16.1 g (0.1 moles) ofL-carnitine inner salt were dissolved in 500 mL of water.

To the resulting solution, 11.6 g (0.1 moles) of fumaric acid were addedunder stirring. Following complete dissolution isobutanol was added andthe mixture distilled under vacuum at 40° C. The residue which wasobtained was taken up with acetone and the mixture left under stirringfor some hours.

The mixture was then filtered under vacuum and the solid thus obtainedwas dried in a thermostatic oven at 30° C. overnight. 40.5 g ofL-carnitine and creatine fumarate were obtained as a white, crystallinesolid which proved to be non-hygroscopic and of pleasant taste.

Yield 96%. m.p. = 134° C. (dec.) K.F. = 0.7% [α]_(D) ²⁰ = −10.7 (c = 1%H₂O) pH = 5.5 (c = 1% H₂O) Ratio: L-carnitine 40% Creatine 32% Fumaricacid 28% Elementary analysis C % H % N % Calculated 44.22 6.67 13.75Found 44.01 6.59 13.68

3.9(2H, s, N—CH ₂—COOH); 3.4-3.3(2H, d, N—CH ₂—CH); 3.2(9H, s, (CH₃)₃—N); 2.9(3H, s, N—CH ₃); 2.5-2.4(2H, d, CH ₂—COOH) HPLC: Column:Hypersil APS-2 (5 μm) 200 × 4.6 Temperature: = =30° C. Mobile phase:CH₃CN/H₂O + 0.05 M KH₂PO₄/CH₃CN (65-35 v/v) pH: 4.7 with H₃PO₄Flow-rate: 0.7 mL/min R_(t) = fumaric acid 12.5; creatine 7.4;L-carnitine 10.8.

EXAMPLE 2 Isovaleryl L-Carnitine and Creatine Fumarate (BS/232

14.9 g (0.1 moles) of creatine monohydrate and 24.5 g (0.1 moles) ofisovaleryl L-carnitine inner salt were dissolved in 500 mL of water.

To the resulting solution 11.6 g (0.1 moles) of fumaric acid were addedunder stirring. Following complete dissolution, isobutanol was added andthe mixture distilled under vacuum at 40° C. The residue thus obtainedwas taken up with acetone and the resulting mixture left under stirringfor some hours.

The mixture was then filtered under vacuum and the solid thus obtaineddried in a thermostatic oven at 30° C. overnight. 47.2 g of isovalerylL-carnitine and creatine fumarate were obtained as a white, crystallinesolid which proved to be non-hygroscopic and could be crystallized from95% ethanol.

Yield: 97% m.p. = 125-127° C. (dec.) K.F. = 0.5% [α]_(D) ²⁰ = −8 (c = 1%H₂O) pH = 5.3 (c = 1% H₂O) Elementary analysis C % H % N % Calculated48.9 7.13 11.4  Found 48.7 7.11 10.98

3.9(2H, s, N—CH ₂—COOH); 3.4-3.3(2H, d, N—CH ₂—CH); 3.2(9H, s, (CH₃)₃—N; 2.9(3H, s, N—CH ₃);

HPLC: Column: Hypersil APS-2 (5 μm) 200 × 4.6 Temperature: = 30° C.Mobile phase: CH₃CN/H₂O + 0.05 M KH₂PO₄/CH₃CN (65-35 v/v) pH: 4.7 withH₃PO₄ Flow-rate: 0.7 mL/min λ: 205 nm R_(t) = fumaric acid 12.5;creatine 7.4; isovaleryl L-carnitine 6.3. Ratio: Isovaleryl 50%L-carnitine Creatine 27% Fumaric acid 23%.

EXAMPLE 3 L-Carnitine and L-Ornithine Fumarate (BS/238)

8 g (0.05 moles) of L-carnitine inner salt, 5.8 g (0.05 moles) offumaric acid and 6.6 g of L-ornithine were dissolved in 7.5 mL of waterat 60° C. and the resulting thick, clear mass was slowly poured into asolution of acetone (800 mL) under vigorous mechanical stirring. A solidprecipitated which was filtered off and dried. 17 g of the titlecompound as a white non-hygroscopic solid were obtained.

Yield: 92% m.p. = 185-187° C. (dec.) K.F. = 0.9% [α]_(D) ²⁰ = −7.5 (c =1% H₂O) pH = 4.7 NMR: D₂O = 6.6 (2H, s, CH═CH); 4.6-4.4 (1H, m, —CH—);3.8-3.6 (1H, t, —CH—NH₂); 3.4-3.3 (2H,d, N—CH ₂); 3.2 (9H, s, (CH ₃)₃);3-2.9 (2H, t, CH ₂—NH₂); 2.6-2.5 (2H, d, —CH ₂—COOH); 2-1.8 (2H, m, CH₂—CH₂—NH₂); 1.8-1.6 (2H, q, CH₂—CH ₂—CH). HPLC: Column: Hypersil APS-2(5 nm) 200 × 4.6 Temperature: = 30° C. Mobile phase: CH₃CN/H₂O + 0.05 MKH₂PO₄/CH₃CN (65-35 v/v) pH: 4.7 with H₃PO₄ Flow-rate: 0.7 mL/min R_(t)= fumaric acid 12.5; L-carnitine 10.8; L-ornithine 9. Ratio: Fumaricacid 28.3% L-carnitine 39.4% L-ornithine 32.3%.

EXAMPLE 4 L-Carnitine and Lysine Fumarate/isovaleryl L-carnitineFumarate (BS/239, BS/240)

Following the procedures of Examples 1 and 2 and substituting 0.1 molesof lysine for 0.1 moles of creatine monohydrate, L-carnitine and lysinefumarate and, respectively, isovaleryl L-carnitine and lysine fumarateoccurring as white non-hygroscopic compounds were prepared.

In the following Table 1 the weight increase (%) and the appearance ofsome compounds of the present invention are shown in comparison withL-carnitine and isovaleryl L-carnitine inner salts and anhydrouscreatine after exposure of the compounds to a relative humidity of 60±5% at 25%, for 24 hours.

Reference: Pharmaeuropa, November 1996.

Table 1 Compound Weight increase % Appearance L-carnitine inner salt 19deliquescent Isovaleryl L-carnitine inner salt 20 deliquescent Anhydrouscreatine 3 flowable Compound of Ex. 1 (BS/231) 0.18 no variationCompound of Ex. 2 (BS/232) 0.19 no variation Compound of Ex. 3 (BS/238)0.16 no variation

The preparation of compositions containing at least one of the doublefumarates of formula (I) shall be readily apparent to any expert inpharmaceutical technology or pharmacy.

The compositions may further contain other ingredients such asantioxidants, coenzymes and mineral substances and may occur in the formof tablets, chewable tablets, pills, troches, lozenges, capsules,granulates or powders.

In unit dosage form, they may contain an amount of a fumarate of formula(I) providing 50-2000, preferably 100-1000, mg of L-carnitine orisovaleryl L-carnitine as inner salt.

What is claimed is:
 1. A fumarate of formula (I):

wherein R is hydrogen or isovaleryl and [A⁺] is a positively chargedamino acid selected from the group consisting of creatine, ornithine,lysine, arginine, and histidine.
 2. The fumarate of claim 1, selectedfrom the group consisting of: L-carnitine and creatine fumarate;isovaleryl L-carnitine and creatine fumarate; L-carnitine and ornithinefumarate; isovaleryl L-carnitine and ornithine fumarate; L-carnitine andlysine fumarate; isovaleryl L-carnitine and lysine fumarate; L-carnitineand arginine fumarate; isovaleryl L-carnitine and arginine fumarate;L-carnitine and histidine fumarate; and isovaleryl L-carnitine andhystidine fumarate.
 3. A composition of a fumarate of general formula(I) as claimed in claim 1 as active principle and a pharmacologicallyacceptable excipient.
 4. The composition of claim 3, further comprisingone or more other ingredients selected from antioxidants, coenzymes andminerals.
 5. The composition of claim 3, in the form of tablets,chewable tablets, pills, troches, lozenges, capsules, granulates orpowders.
 6. The composition of claim 3, in unit dosage form, comprisingas active ingredient a fumarate of formula (I) containing 50-2000,preferably 100-1000, mg of L-carnitine or isovaleryl L-carnitine asinner salt.
 7. The composition of claim 3, for human use as foodsupplement, dietary supplement or a drug.
 8. The composition of claim 3,for veterinary use as supplement for fodders.